Vibrio cholerae, a gram-negative halophilic bacterium found in aquatic environments, is the causative agent of the epidemic diarrheal disease cholera. Because V. cholerae is primarily an environmental organism rather than a human pathogen, many have speculated that V. cholerae virulence factors must also target an as yet unidentified non-human host. Insects have previously been implicated in the environmental spread of V. cholerae, and, in areas where cholera is endemic, several reports exist of isolation of V. cholerae from Musca domestica, the common house fly. Based on these findings, we conducted preliminary experiments exploring the novel hypotheses that insects that are fed V. cholerae develop a disease similar to cholera and that insects may be used as model organisms in which to study the human disease. To test these hypotheses, we developed a model of oral infection using Drosophila melanogaster, an intensely studied and well-developed model organism. In this R21 proposal, we will test the hypothesis that the path of V. cholerae through the fly, cholera toxin trafficking, and the mechanism of action of cholera toxin within Drosophila cells are similar to that in humans. We would also like to demonstrate the existence of both known and novel V. cholerae and Drosophila genes that define the course of infection. This will lay the groundwork for future studies using the fly as a model to better understand the interplay between V. cholerae and its host. The following specific aims are proposed: Specific Aim 1: To spatially and temporally map the physical path and transcriptional response of V. cholerae following ingestion by the fly. Specific Aim 2: To identify Drosophila genes that alter the course of V. cholerae infection.